Difference between revisions of "Part:BBa K2361000:Design"
(→‎Design Notes)
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
This is Cas9 with two amino acid substitutions (D10A & H840A) that disrupt its two nuclease domains. If the normal variant was used, it would cut up our reporter plasmid. This was the initial starting protein as the modifications were made later to target specific sequences related to the Groningen 2017 project and to meet all the biobrick characteristics.
+
This part originates from <i>Streptococcus pyogenes</i> Cas9 and it contains two amino acid substitutions (D10A & H840A) that make it catalytically dead. Also it contains one base substitution (T->A) that remove the illegal EcoRI site. The two amino acid substitutions were already present in the part we started with, so we made it biobrick compatible by removing the EcoRI site and adding the prefix and suffix.
−
 
+
−
 
+
  
 
===Source===
 
===Source===

Revision as of 09:44, 27 October 2017


spdCas9


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1100
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3379
    Illegal XhoI site found at 4115
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part originates from Streptococcus pyogenes Cas9 and it contains two amino acid substitutions (D10A & H840A) that make it catalytically dead. Also it contains one base substitution (T->A) that remove the illegal EcoRI site. The two amino acid substitutions were already present in the part we started with, so we made it biobrick compatible by removing the EcoRI site and adding the prefix and suffix.

Source

This part originates from the Addgene plasmid pJWV102-PL-dCas9. PCR was used to amplify the protein coding sequence to integrate into a biobrick format.

References

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW. Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.